Publications by authors named "Ankit Sinha"

35 Publications

Mammary epithelial cells have lineage-rooted metabolic identities.

Nat Metab 2021 May 20;3(5):665-681. Epub 2021 May 20.

Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

Cancer metabolism adapts the metabolic network of its tissue of origin. However, breast cancer is not a disease of a single origin. Multiple epithelial populations serve as the culprit cell of origin for specific breast cancer subtypes, yet our knowledge of the metabolic network of normal mammary epithelial cells is limited. Using a multi-omic approach, here we identify the diverse metabolic programmes operating in normal mammary populations. The proteomes of basal, luminal progenitor and mature luminal cell populations revealed enrichment of glycolysis in basal cells and of oxidative phosphorylation in luminal progenitors. Single-cell transcriptomes corroborated lineage-specific metabolic identities and additional intra-lineage heterogeneity. Mitochondrial form and function differed across lineages, with clonogenicity correlating with mitochondrial activity. Targeting oxidative phosphorylation and glycolysis with inhibitors exposed lineage-rooted metabolic vulnerabilities of mammary progenitors. Bioinformatics indicated breast cancer subtypes retain metabolic features of their putative cell of origin. Thus, lineage-rooted metabolic identities of normal mammary cells may underlie breast cancer metabolic heterogeneity and targeting these vulnerabilities could advance breast cancer therapy.
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http://dx.doi.org/10.1038/s42255-021-00388-6DOI Listing
May 2021

Comment on 'Neuroradiology for ophthalmologists'.

Eye (Lond) 2021 Apr 6. Epub 2021 Apr 6.

Department of Ophthalmology, National Hospital for Neurology and Neurosurgery, London, UK.

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http://dx.doi.org/10.1038/s41433-021-01406-3DOI Listing
April 2021

NASH limits anti-tumour surveillance in immunotherapy-treated HCC.

Nature 2021 Apr 24;592(7854):450-456. Epub 2021 Mar 24.

Translational and Clinical Research Institute, Faculty of Medical Sciences, Newcastle University, Newcastle, UK.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need. Here we report the progressive accumulation of exhausted, unconventionally activated CD8PD1 T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8PD1 T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8PD1CXCR6, TOX, and TNF T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8 T cells or TNF neutralization, suggesting that CD8 T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8PD1 T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.
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http://dx.doi.org/10.1038/s41586-021-03362-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8046670PMC
April 2021

Ethnicity and COVID-19 - A commentary on "World Health Oganization declares global emergency: A review of the 2019 novel coronavirus (COVID-19)" (Int J Surg 2020;76:71-6).

Int J Surg 2020 11 11;83:75-76. Epub 2020 Sep 11.

University College London Medical School, 74 Huntley Street, Bloomsbury, London, WC1E 6DE, UK.

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http://dx.doi.org/10.1016/j.ijsu.2020.08.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484806PMC
November 2020

Magnetic resonance imaging reveals specific anatomical changes in the brain of Agat- and Gamt-mice attributed to creatine depletion and guanidinoacetate alteration.

J Inherit Metab Dis 2020 07 27;43(4):827-842. Epub 2020 Jan 27.

Genetics and Genome Biology Program, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.

Arginine:glycine amidinotransferase- and guanidinoacetate methyltransferase deficiency are severe neurodevelopmental disorders. It is not known whether mouse models of disease express a neuroanatomical phenotype. High-resolution magnetic resonance imaging (MRI) with advanced image analysis was performed in perfused, fixed mouse brains encapsulated with the skull from male, 10-12 week old Agat and B6J.Cg-Gamt mice (n = 48; n = 8 per genotype, strain). T2-weighted MRI scans were nonlinearly aligned to a 3D atlas of the mouse brain with 62 structures identified. Local differences in brain shape related to genotype were assessed by analysis of deformation fields. Creatine (Cr) and guanidinoacetate (GAA) were measured with high-performance liquid chromatography (HPLC) in brain homogenates (n = 24; n = 4 per genotype, strain) after whole-body perfusion. Cr was decreased in the brain of Agat- and Gamt mutant mice. GAA was decreased in Agat and increased in Gamt . Body weight and brain volume were lower in Agat than in Gamt . The analysis of entire brain structures revealed corpus callosum, internal capsule, fimbria and hypothalamus being different between the genotypes in both strains. Eighteen and fourteen significant peaks (local areas of difference in relative size) were found in Agat- and Gamt mutants, respectively. Comparing Agat with Gamt , we found changes in three brain regions, lateral septum, amygdala, and medulla. Intra-strain differences in four brain structures can be associated with Cr deficiency, while the inter-strain differences in three brain structures of the mutant mice may relate to GAA. Correlating these neuroanatomical findings with gene expression data implies the role of Cr metabolism in the developing brain and the importance of early intervention in patients with Cr deficiency syndromes.
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http://dx.doi.org/10.1002/jimd.12215DOI Listing
July 2020

Dissecting intercellular signaling with mass spectrometry-based proteomics.

Curr Opin Cell Biol 2020 04 9;63:20-30. Epub 2020 Jan 9.

Experimental Systems Immunology Laboratory, Max-Planck Institute of Biochemistry, Martinsried, Germany. Electronic address:

Physiological functions depend on a coordinated interplay of numerous different cell types. Proteins serve as major signaling molecules between cells; however, their comprehensive investigation in physiologically relevant settings has remained challenging. Mass spectrometry (MS)-based shotgun proteomics is emerging as a powerful technology for the systematic analysis of protein-mediated intercellular signaling and regulated post-translational modifications. Here, we discuss recent advancements in cell biological, chemical, and biochemical MS-based approaches for the profiling of cellular messengers released by sending cells, receptors expressed on the cell surface, and their interactions. We highlight methods tailored toward the mapping of dynamic signal transduction mechanisms at cellular interfaces and approaches to dissect communication cell specifically in heterocellular systems. Thereby, MS-based proteomics contributes a unique systems biology perspective for the identification of intercellular signaling pathways deregulated in disease.
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http://dx.doi.org/10.1016/j.ceb.2019.12.002DOI Listing
April 2020

Neuroleptic malignant syndrome associated with the use of injection zuclopenthixol acetate.

Ind Psychiatry J 2020 Jan-Jun;29(1):162-164. Epub 2020 Nov 7.

Department of Psychiatry, Postgraduate Institute of Medical Education and Research, Chandigarh, India.

Zuclopenthixol is available in two parenteral formulation,i.e., in the form of acetate and decanoate. It has high affinity for dopamine D1 and D2 receptors. There is limited literature of association of neuroleptic malignant syndrome with the use of zuclopenthixol monotherapy. These case reports have mostly implicated zuclopenthixol decanoate, and also zuclopenthixol acetate. In this report, we present a case of neuroleptic malignant syndrome associated with use of zuclopenthixol acetate.
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http://dx.doi.org/10.4103/ipj.ipj_54_19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7989464PMC
November 2020

Genome-wide germline correlates of the epigenetic landscape of prostate cancer.

Nat Med 2019 10 7;25(10):1615-1626. Epub 2019 Oct 7.

Ontario Institute for Cancer Research, Toronto, Ontario, Canada.

Oncogenesis is driven by germline, environmental and stochastic factors. It is unknown how these interact to produce the molecular phenotypes of tumors. We therefore quantified the influence of germline polymorphisms on the somatic epigenome of 589 localized prostate tumors. Predisposition risk loci influence a tumor's epigenome, uncovering a mechanism for cancer susceptibility. We identified and validated 1,178 loci associated with altered methylation in tumoral but not nonmalignant tissue. These tumor methylation quantitative trait loci influence chromatin structure, as well as RNA and protein abundance. One prominent tumor methylation quantitative trait locus is associated with AKT1 expression and is predictive of relapse after definitive local therapy in both discovery and validation cohorts. These data reveal intricate crosstalk between the germ line and the epigenome of primary tumors, which may help identify germline biomarkers of aggressive disease to aid patient triage and optimize the use of more invasive or expensive diagnostic assays.
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http://dx.doi.org/10.1038/s41591-019-0579-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418214PMC
October 2019

Age-Related Gliosis Promotes Central Nervous System Lymphoma through CCL19-Mediated Tumor Cell Retention.

Cancer Cell 2019 09;36(3):250-267.e9

Department of Pathology and Molecular Pathology, University Hospital of Zurich, 8091 Zurich, Switzerland.

How lymphoma cells (LCs) invade the brain during the development of central nervous system lymphoma (CNSL) is unclear. We found that NF-κB-induced gliosis promotes CNSL in immunocompetent mice. Gliosis elevated cell-adhesion molecules, which increased LCs in the brain but was insufficient to induce CNSL. Astrocyte-derived CCL19 was required for gliosis-induced CNSL. Deleting CCL19 in mice or CCR7 from LCs abrogated CNSL development. Two-photon microscopy revealed LCs transiently entering normal brain parenchyma. Astrocytic CCL19 enhanced parenchymal CNS retention of LCs, thereby promoting CNSL formation. Aged, gliotic wild-type mice were more susceptible to forming CNSL than young wild-type mice, and astrocytic CCL19 was observed in both human gliosis and CNSL. Therefore, CCL19-CCR7 interactions may underlie an increased age-related risk for CNSL.
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http://dx.doi.org/10.1016/j.ccell.2019.08.001DOI Listing
September 2019

Metalloprotease inhibitor TIMP proteins control FGF-2 bioavailability and regulate skeletal growth.

J Cell Biol 2019 09 1;218(9):3134-3152. Epub 2019 Aug 1.

Princess Margaret Cancer Centre/Ontario Cancer Institute, University Health Network, Toronto, Canada

Regulated growth plate activity is essential for postnatal bone development and body stature, yet the systems regulating epiphyseal fusion are poorly understood. Here, we show that the tissue inhibitors of metalloprotease (TIMP) gene family is essential for normal bone growth after birth. Whole-body quadruple-knockout mice lacking all four TIMPs have growth plate closure in long bones, precipitating limb shortening, epiphyseal distortion, and widespread chondrodysplasia. We identify TIMP/FGF-2/IHH as a novel nexus underlying bone lengthening where TIMPs negatively regulate the release of FGF-2 from chondrocytes to allow IHH expression. Using a knock-in approach that combines MMP-resistant or ADAMTS-resistant aggrecans with TIMP deficiency, we uncouple growth plate activity in axial and appendicular bones. Thus, natural metalloprotease inhibitors are crucial regulators of chondrocyte maturation program, growth plate integrity, and skeletal proportionality. Furthermore, individual and combinatorial TIMP-deficient mice demonstrate the redundancy of metalloprotease inhibitor function in embryonic and postnatal development.
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http://dx.doi.org/10.1083/jcb.201906059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719459PMC
September 2019

N-Glycoproteomics of Patient-Derived Xenografts: A Strategy to Discover Tumor-Associated Proteins in High-Grade Serous Ovarian Cancer.

Cell Syst 2019 04 10;8(4):345-351.e4. Epub 2019 Apr 10.

University of Toronto, Department of Medical Biophysics, Toronto, ON M5G 1L7, Canada; Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 2M9, Canada. Electronic address:

High-grade serous ovarian carcinoma (HGSC) is the most common and lethal subtype of gynecologic malignancy in women. The current standard of treatment combines cytoreductive surgery and chemotherapy. Despite the efficacy of initial treatment, most patients develop cancer recurrence, and 70% of patients die within 5 years of initial diagnosis. CA125 is the current FDA-approved biomarker used in the clinic to monitor response to treatment and recurrence, but its impact on patient survival is limited. New strategies for the discovery of HGSC biomarkers are urgently needed. Here, we describe a proteomics strategy to detect tumor-associated proteins in serum of HGSC patient-derived xenograft models. We demonstrate proof-of-concept applicability using two independent, longitudinal serum cohorts from HGSC patients.
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http://dx.doi.org/10.1016/j.cels.2019.03.011DOI Listing
April 2019

The Proteogenomic Landscape of Curable Prostate Cancer.

Cancer Cell 2019 03;35(3):414-427.e6

Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada; Ontario Institute for Cancer Research, Toronto, ON M5G 0A3, Canada; Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON M5S 1A8, Canada; Department of Human Genetics, University of California, 12-109 CHS, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA; Department of Urology, University of California, Los Angeles, CA 90024, USA; Jonsson Comprehensive Cancer Centre, University of California, Los Angeles, CA 90024, USA; Institute for Precision Health, University of California, Los Angeles, CA 90024, USA. Electronic address:

DNA sequencing has identified recurrent mutations that drive the aggressiveness of prostate cancers. Surprisingly, the influence of genomic, epigenomic, and transcriptomic dysregulation on the tumor proteome remains poorly understood. We profiled the genomes, epigenomes, transcriptomes, and proteomes of 76 localized, intermediate-risk prostate cancers. We discovered that the genomic subtypes of prostate cancer converge on five proteomic subtypes, with distinct clinical trajectories. ETS fusions, the most common alteration in prostate tumors, affect different genes and pathways in the proteome and transcriptome. Globally, mRNA abundance changes explain only ∼10% of protein abundance variability. As a result, prognostic biomarkers combining genomic or epigenomic features with proteomic ones significantly outperform biomarkers comprised of a single data type.
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http://dx.doi.org/10.1016/j.ccell.2019.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511374PMC
March 2019

Mammary molecular portraits reveal lineage-specific features and progenitor cell vulnerabilities.

J Cell Biol 2018 08 19;217(8):2951-2974. Epub 2018 Jun 19.

Princess Margaret Cancer Centre, Toronto, ON, Canada

The mammary epithelium depends on specific lineages and their stem and progenitor function to accommodate hormone-triggered physiological demands in the adult female. Perturbations of these lineages underpin breast cancer risk, yet our understanding of normal mammary cell composition is incomplete. Here, we build a multimodal resource for the adult gland through comprehensive profiling of primary cell epigenomes, transcriptomes, and proteomes. We define systems-level relationships between chromatin-DNA-RNA-protein states, identify lineage-specific DNA methylation of transcription factor binding sites, and pinpoint proteins underlying progesterone responsiveness. Comparative proteomics of estrogen and progesterone receptor-positive and -negative cell populations, extensive target validation, and drug testing lead to discovery of stem and progenitor cell vulnerabilities. Top epigenetic drugs exert cytostatic effects; prevent adult mammary cell expansion, clonogenicity, and mammopoiesis; and deplete stem cell frequency. Select drugs also abrogate human breast progenitor cell activity in normal and high-risk patient samples. This integrative computational and functional study provides fundamental insight into mammary lineage and stem cell biology.
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http://dx.doi.org/10.1083/jcb.201804042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080920PMC
August 2018

Proteomic Analysis of Cancer-Associated Fibroblasts Reveals a Paracrine Role for MFAP5 in Human Oral Tongue Squamous Cell Carcinoma.

J Proteome Res 2018 06 2;17(6):2045-2059. Epub 2018 May 2.

Princess Margaret Cancer Centre , University Health Network , Toronto , Ontario M5G 1L7 , Canada.

Bidirectional communication between cells and their microenvironment is crucial for both normal tissue homeostasis and tumor growth. During the development of oral tongue squamous cell carcinoma (OTSCC), cancer-associated fibroblasts (CAFs) create a supporting niche by maintaining a bidirectional crosstalk with cancer cells, mediated by classically secreted factors and various nanometer-sized vesicles, termed as extracellular vesicles (EVs). To better understand the role of CAFs within the tumor stroma and elucidate the mechanism by which secreted proteins contribute to OTSCC progression, we isolated and characterized patient-derived CAFs from resected tumors with matched adjacent tissue fibroblasts (AFs). Our strategy employed shotgun proteomics to comprehensively characterize the proteomes of these matched fibroblast populations. Our goals were to identify CAF-secreted factors (EVs and soluble) that can functionally modulate OTSCC cells in vitro and to identify novel CAF-associated biomarkers. Comprehensive proteomic analysis identified 4247 proteins, the most detailed description of a pro-tumorigenic stroma to date. We demonstrated functional effects of CAF secretomes (EVs and conditioned media) on OTSCC cell growth and migration. Comparative proteomics identified novel proteins associated with a CAF-like state. Specifically, MFAP5, a protein component of extracellular microfibrils, was enriched in CAF secretomes. Using in vitro assays, we demonstrated that MFAP5 activated OTSCC cell growth and migration via activation of MAPK and AKT pathways. Using a tissue microarray of richly annotated primary human OTSCCs, we demonstrated an association of MFAP5 expression with patient survival. In summary, our proteomics data of patient-derived stromal fibroblasts provide a useful resource for future mechanistic and biomarker studies.
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http://dx.doi.org/10.1021/acs.jproteome.7b00925DOI Listing
June 2018

Proteomic Profiling of Secreted Proteins, Exosomes, and Microvesicles in Cell Culture Conditioned Media.

Methods Mol Biol 2018 ;1722:91-102

Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada.

Secreted proteins are of tremendous biological interest since they can act as ligands for receptors to activate downstream signalling cascades or be used as biomarkers if altered abundance is correlated with a specific pathological state. Proteins can be secreted either as soluble molecules or as part of extracellular vesicles (i.e., exosomes or microvesicles). The complete proteomic profiling of secretomes requires analysis of secreted proteins and extracellular vesicles. Hence, the method described here enriches for microvesicles, exosomes, and secreted proteins from conditioned media using differential centrifugation. The three fractions are then analyzed by mass spectrometry-based proteomics for in-depth characterization and comparison of the protein secretome of cell lines.
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http://dx.doi.org/10.1007/978-1-4939-7553-2_6DOI Listing
August 2018

Divergent evolution of temozolomide resistance in glioblastoma stem cells is reflected in extracellular vesicles and coupled with radiosensitization.

Neuro Oncol 2018 01;20(2):236-248

McGill University, Research Institute of the McGill University Health Centre (RIMUHC), Montreal, Quebec, Canada.

Background: Glioblastoma (GBM) is almost invariably fatal due to failure of standard therapy. The relapse of GBM following surgery, radiation, and systemic temozolomide (TMZ) is attributed to the ability of glioma stem cells (GSCs) to survive, evolve, and repopulate the tumor mass, events on which therapy exerts a poorly understood influence.

Methods: Here we explore the molecular and cellular evolution of TMZ resistance as it emerges in vivo (xenograft models) in a series of human GSCs with either proneural (PN) or mesenchymal (MES) molecular characteristics.

Results: We observed that the initial response of GSC-initiated intracranial xenografts to TMZ is eventually replaced by refractory growth pattern. Individual tumors derived from the same isogenic GSC line expressed divergent and complex profiles of TMZ resistance markers, with a minor representation of O6-methylguanine DNA methyltransferase (MGMT) upregulation. In several independent TMZ-resistant tumors originating from MES GSCs we observed a consistent diminution of mesenchymal features, which persisted in cell culture and correlated with increased expression of Nestin, decline in transglutaminase 2 and sensitivity to radiation. The corresponding mRNA expression profiles reflective of TMZ resistance and stem cell phenotype were recapitulated in the transcriptome of exosome-like extracellular vesicles (EVs) released by GSCs into the culture medium.

Conclusions: Intrinsic changes in the tumor-initiating cell compartment may include loss of subtype characteristics and reciprocal alterations in sensitivity to chemo- and radiation therapy. These observations suggest that exploiting therapy-induced changes in the GSC phenotype and alternating cycles of therapy may be explored to improve GBM outcomes.
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http://dx.doi.org/10.1093/neuonc/nox142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777501PMC
January 2018

Proteomic Response of Human Umbilical Vein Endothelial Cells to Histamine Stimulation.

Proteomics 2017 Nov 12;17(21). Epub 2017 Oct 12.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.

The histamine receptors (HRs) represent a subclass of G protein-coupled receptors (GPCRs) and comprise four subtypes. Due to their numerous physiological and pathological effects, HRs are popular drug targets for the treatment of allergic reactions or the regulation of gastric acid secretion. Hence, an understanding of the functional selectivity of HR ligands has gained importance. These ligands can bind to specific GPCRs and selectively activate defined pathways. Supporting the activation of a therapeutically necessary pathway without the activation of other signaling cascades can result in drugs with more specific activity and fewer side effects. To evaluate the cellular consequences resulting from receptor binding, comprehensive analyses of cellular protein alterations upon incubation with ligands are required. For this purpose, endothelial cells are treated with histamine, as the endogenous ligand of HRs, to obtain a global overview of its cellular effects. Quantitative proteomics and pathway analyses of histamine-treated and untreated cells reveal enrichment of the nuclear factor-κB and tumor necrosis factor signaling pathways, cytokine-cytokine receptor interactions, complement and coagulation cascades, and acute inflammatory processes upon histamine treatment. This strategy offers the opportunity to monitor HR-mediated signaling in a multidimensional manner.
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http://dx.doi.org/10.1002/pmic.201700116DOI Listing
November 2017

Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors.

Nat Commun 2017 08 24;8(1):331. Epub 2017 Aug 24.

Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada, M5G 1L7.

PDX1/NKX6-1 pancreatic progenitors (PPs) give rise to endocrine cells both in vitro and in vivo. This cell population can be successfully differentiated from human pluripotent stem cells (hPSCs) and hold the potential to generate an unlimited supply of β cells for diabetes treatment. However, the efficiency of PP generation in vitro is highly variable, negatively impacting reproducibility and validation of in vitro and in vivo studies, and consequently, translation to the clinic. Here, we report the use of a proteomics approach to phenotypically characterize hPSC-derived PPs and distinguish these cells from non-PP populations during differentiation. Our analysis identifies the pancreatic secretory granule membrane major glycoprotein 2 (GP2) as a PP-specific cell surface marker. Remarkably, GP2 is co-expressed with NKX6-1 and PTF1A in human developing pancreata, indicating that it marks the multipotent pancreatic progenitors in vivo. Finally, we show that isolated hPSC-derived GP2 cells generate β-like cells (C-PEPTIDE/NKX6-1) more efficiently compared to GP2 and unsorted populations, underlining the potential therapeutic applications of GP2.Pancreatic progenitors (PPs) can be derived from human pluripotent stem cells in vitro but efficiency of differentiation varies, making it hard to sort for insulin-producing cells. Here, the authors use a proteomic approach to identify the secretory granule membrane glycoprotein 2 as a marker for PDX1+/NKX6-1+ PPs.
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http://dx.doi.org/10.1038/s41467-017-00561-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5569081PMC
August 2017

Detecting protein variants by mass spectrometry: a comprehensive study in cancer cell-lines.

Genome Med 2017 07 18;9(1):62. Epub 2017 Jul 18.

Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.

Background: Onco-proteogenomics aims to understand how changes in a cancer's genome influences its proteome. One challenge in integrating these molecular data is the identification of aberrant protein products from mass-spectrometry (MS) datasets, as traditional proteomic analyses only identify proteins from a reference sequence database.

Methods: We established proteomic workflows to detect peptide variants within MS datasets. We used a combination of publicly available population variants (dbSNP and UniProt) and somatic variations in cancer (COSMIC) along with sample-specific genomic and transcriptomic data to examine proteome variation within and across 59 cancer cell-lines.

Results: We developed a set of recommendations for the detection of variants using three search algorithms, a split target-decoy approach for FDR estimation, and multiple post-search filters. We examined 7.3 million unique variant tryptic peptides not found within any reference proteome and identified 4771 mutations corresponding to somatic and germline deviations from reference proteomes in 2200 genes among the NCI60 cell-line proteomes.

Conclusions: We discuss in detail the technical and computational challenges in identifying variant peptides by MS and show that uncovering these variants allows the identification of druggable mutations within important cancer genes.
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http://dx.doi.org/10.1186/s13073-017-0454-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5514513PMC
July 2017

HLA-DP constitutively presents endogenous peptides generated by the class I antigen processing pathway.

Nat Commun 2017 05 10;8:15244. Epub 2017 May 10.

Tumor Immunotherapy Program, Campbell Family Institute for Breast Cancer Research, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada M5G 2M9.

Classical antigen processing leads to the presentation of antigenic peptides derived from endogenous and exogenous sources for MHC class I and class II molecules, respectively. Here we show that, unlike other class II molecules, prevalent HLA-DP molecules with β-chains encoding Gly84 (DP) constitutively present endogenous peptides. DP does not bind invariant chain (Ii) via the class II-associated invariant chain peptide (CLIP) region, nor does it present CLIP. However, Ii does facilitate the transport of DP from the endoplasmic reticulum (ER) to the endosomal/lysosomal pathway by transiently binding DP via a non-CLIP region(s) in a pH-sensitive manner. Accordingly, like class I, DP constitutively presents endogenous peptides processed by the proteasome and transported to the ER by the transporter associated with antigen processing (TAP). Therefore, DP, found only in common chimpanzees and humans, uniquely uses both class I and II antigen-processing pathways to present peptides derived from intracellular and extracellular sources.
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http://dx.doi.org/10.1038/ncomms15244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5436232PMC
May 2017

Characterization of Protein Content Present in Exosomes Isolated from Conditioned Media and Urine.

Curr Protoc Protein Sci 2017 02 2;87:24.9.1-24.9.12. Epub 2017 Feb 2.

University of Toronto, Department of Medical Biophysics, Toronto, Ontario, Canada.

Cells secrete biomolecules into the extracellular space as a way of intercellular communication. Secreted proteins can act as ligands that engage specific receptors-on the same cell, nearby cells, or distant cells-and induce defined signaling pathways. Proteins and other biomolecules can also be packaged as cargo molecules within vesicles that are released to the extracellular space (termed extracellular vesicles or EVs). A subclass of such EVs, exosomes have been shown to horizontally transfer information. In recent years, exosomes have sparked tremendous interest in biological research, both for the discovery of novel biomarkers and for the identification of signaling molecules, as part of their cargo. Although multiple methods have been described for the isolation of exosomes, described here is a simple differential centrifugation approach that is well suited for the isolation of exosomes from conditioned cell culture media and urine. Mass spectrometry provides an ideal method to comprehensively analyze the protein cargo of exosomes. © 2017 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpps.23DOI Listing
February 2017

Primary pleural leiomyosarcoma - A rare entity.

Lung India 2017 Jan-Feb;34(1):104-105

Department of Pathology, Metro Multi Specialty Hospital, Noida Sector 11, Uttar Pradesh, India.

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http://dx.doi.org/10.4103/0970-2113.197095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5234184PMC
February 2017

march: from ABPA to aspergilloma to subacute invasive aspergillosis.

Allergy Asthma Clin Immunol 2016 3;12:64. Epub 2016 Dec 3.

Metro Center for Respiratory Diseases, Metro Multispeciality Hospital, Sector 11, Noida, Uttar Pradesh India.

Background: is a ubiquitous fungus responsible for allergic as well as saprophytic and invasive manifestations depending on host's immune status. The following case report demonstrates progression of allergic manifestations of to its invasive form in an individual with decreasing immunity. This can lead to uncertainties in diagnosis and management.

Case Presentation: A 28-year-old male, non smoker, known case of ABPA (allergic bronchopulmonary aspergillosis) was admitted with complaints of cough for 1 month, associated with recurrent episodes of hemoptysis for last 5 days. CT Thorax revealed homogenous dense round opacity in right upper lobe which replaced previous fibrocalcific bronchiectatic lesion with cavity and aspergilloma, bulging across the major fissure with fibrotic strands extending to periphery in all directions. Post-pneumonectomy microscopic examination revealed hyphae invading blood vessels.

Conclusion: There is a need for close clinical and radiologic follow up of patients with and our patient demonstrated overlap of complete spectrum of disease with march from one end to the other end.
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http://dx.doi.org/10.1186/s13223-016-0170-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5135745PMC
December 2016

Postpericardiotomy syndrome: What a pulmonologist must know.

Lung India 2016 Sep-Oct;33(5):586-7

Metro Centre for Respiratory Diseases, Metro Multispeciality Hospital, Noida, Uttar Pradesh, India E-mail:

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http://dx.doi.org/10.4103/0970-2113.189010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006357PMC
September 2016

Ectopic miR-125a Expression Induces Long-Term Repopulating Stem Cell Capacity in Mouse and Human Hematopoietic Progenitors.

Cell Stem Cell 2016 09 14;19(3):383-96. Epub 2016 Jul 14.

Laboratory of Ageing Biology and Stem Cells, European Research Institute for the Biology of Ageing, University Medical Centre Groningen, University of Groningen, Antonius Deusinglaan 1, 9700 AV Groningen, the Netherlands. Electronic address:

Umbilical cord blood (CB) is a convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. Although one feasible option would be to expand HSCs to improve therapeutic outcome, available protocols and the molecular mechanisms governing the self-renewal of HSCs are unclear. Here, we show that ectopic expression of a single microRNA (miRNA), miR-125a, in purified murine and human multipotent progenitors (MPPs) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and western blot analysis, we identified a restricted set of miR-125a targets involved in conferring long-term repopulating capacity to MPPs in humans and mice. Our findings offer the innovative potential to use MPPs with enhanced self-renewal activity to augment limited sources of HSCs to improve clinical protocols.
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http://dx.doi.org/10.1016/j.stem.2016.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500905PMC
September 2016

PTP1B controls non-mitochondrial oxygen consumption by regulating RNF213 to promote tumour survival during hypoxia.

Nat Cell Biol 2016 07 20;18(7):803-813. Epub 2016 Jun 20.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 1L7, Canada.

Tumours exist in a hypoxic microenvironment and must limit excessive oxygen consumption. Hypoxia-inducible factor (HIF) controls mitochondrial oxygen consumption, but how/if tumours regulate non-mitochondrial oxygen consumption (NMOC) is unknown. Protein-tyrosine phosphatase-1B (PTP1B) is required for Her2/Neu-driven breast cancer (BC) in mice, although the underlying mechanism and human relevance remain unclear. We found that PTP1B-deficient HER2(+) xenografts have increased hypoxia, necrosis and impaired growth. In vitro, PTP1B deficiency sensitizes HER2(+) BC lines to hypoxia by increasing NMOC by α-KG-dependent dioxygenases (α-KGDDs). The moyamoya disease gene product RNF213, an E3 ligase, is negatively regulated by PTP1B in HER2(+) BC cells. RNF213 knockdown reverses the effects of PTP1B deficiency on α-KGDDs, NMOC and hypoxia-induced death of HER2(+) BC cells, and partially restores tumorigenicity. We conclude that PTP1B acts via RNF213 to suppress α-KGDD activity and NMOC. This PTP1B/RNF213/α-KGDD pathway is critical for survival of HER2(+) BC, and possibly other malignancies, in the hypoxic tumour microenvironment.
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http://dx.doi.org/10.1038/ncb3376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4936519PMC
July 2016

Expansion of stem cells counteracts age-related mammary regression in compound Timp1/Timp3 null mice.

Nat Cell Biol 2015 Mar 23;17(3):217-27. Epub 2015 Feb 23.

1] Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada [2] Princess Margaret Cancer Centre/University Health Network, Toronto, Ontario M5G 2M9, Canada [3] Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

Age is the primary risk factor for breast cancer in women. Bipotent basal stem cells actively maintain the adult mammary ductal tree, but with age tissues atrophy. We show that cell-extrinsic factors maintain the adult stem cell pool during ageing and dictate tissue stoichiometry. Mammary stem cells spontaneously expand more than 11-fold in virgin adult female mice lacking specific genes for TIMPs, the natural metalloproteinase inhibitors. Compound Timp1/Timp3 null glands exhibit Notch activation and accelerated gestational differentiation. Proteomics of mutant basal cells uncover altered cytoskeletal and extracellular protein repertoires, and we identify aberrant mitotic spindle orientation in these glands, a process that instructs asymmetric cell division and fate. We find that progenitor activity normally declines with age, but enriched stem/progenitor pools prevent tissue regression in Timp mutant mammary glands without affecting carcinogen-induced cancer susceptibility. Thus, improved stem cell content can extend mouse mammary tissue lifespan without altering cancer risk in this mouse model.
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http://dx.doi.org/10.1038/ncb3118DOI Listing
March 2015

VennDIS: a JavaFX-based Venn and Euler diagram software to generate publication quality figures.

Proteomics 2015 Apr 23;15(7):1239-44. Epub 2015 Jan 23.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.

Venn diagrams are graphical representations of the relationships among multiple sets of objects and are often used to illustrate similarities and differences among genomic and proteomic datasets. All currently existing tools for producing Venn diagrams evince one of two traits; they require expertise in specific statistical software packages (such as R), or lack the flexibility required to produce publication-quality figures. We describe a simple tool that addresses both shortcomings, Venn Diagram Interactive Software (VennDIS), a JavaFX-based solution for producing highly customizable, publication-quality Venn, and Euler diagrams of up to five sets. The strengths of VennDIS are its simple graphical user interface and its large array of customization options, including the ability to modify attributes such as font, style and position of the labels, background color, size of the circle/ellipse, and outline color. It is platform independent and provides real-time visualization of figure modifications. The created figures can be saved as XML files for future modification or exported as high-resolution images for direct use in publications.
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http://dx.doi.org/10.1002/pmic.201400320DOI Listing
April 2015

A rare case: testicular exstrophy or scrotoschisis? A report and discussion.

Pediatr Surg Int 2015 Feb 17;31(2):209-11. Epub 2014 Dec 17.

Department of General Surgery, Burdwan Medical College and Hospital, Burdwan, 713104, West Bengal, India,

Anomalies of testicular descent are very common but scrotal wall deformity leading to extrusion of testes is very rare. This anomaly is described as scrotoschisis or testicular exstrophy. In English literature less than 15 cases were reported till date to the best of our knowledge. A rare case of unilateral testicular exstrophy in a full term 3-day-old neonate is reported here along with review of literature and discussions on probable etiology.
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http://dx.doi.org/10.1007/s00383-014-3650-3DOI Listing
February 2015

Onco-proteogenomics: cancer proteomics joins forces with genomics.

Nat Methods 2014 Nov;11(11):1107-13

1] Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. [2] Informatics &Biocomputing Program, Ontario Institute for Cancer Research, Toronto, Ontario, Canada. [3] Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario, Canada.

The complexities of tumor genomes are rapidly being uncovered, but how they are regulated into functional proteomes remains poorly understood. Standard proteomics workflows use databases of known proteins, but these databases do not capture the uniqueness of the cancer transcriptome, with its point mutations, unusual splice variants and gene fusions. Onco-proteogenomics integrates mass spectrometry-generated data with genomic information to identify tumor-specific peptides. Linking tumor-derived DNA, RNA and protein measurements into a central-dogma perspective has the potential to improve our understanding of cancer biology.
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http://dx.doi.org/10.1038/nmeth.3138DOI Listing
November 2014